The present invention relates generally to the field of delivering agents, including genes and other biological macromolecules, to cells.
The following description of the background of the invention is provided to aid in understanding the claimed invention, but it is not admitted to constitute or describe prior art to the claimed invention and should in no way be construed as limiting the claimed invention.
Several techniques currently exist for delivering genes to cells and many clinical trials are currently ongoing in order to evaluate the degree of therapeutic efficacy obtained using such methods. One method of gene delivery involves the use of recombinant retroviral vectors for delivery of genes to cells of living animals. Morgan et al., Annu. Rev. Biochem., 62:191-217 (1993). Retroviral vectors permanently integrate the transferred gene into the host chromosomal DNA. In addition to retroviruses, other virus have been used for gene delivery. Adenoviruses have been developed as a means for gene transfer into epithelial derived tissues. Stratford-Perricaudet et al., Hum. Gene. Ther., 1:241-256 (1990); Gilardi et al., FEBS, 267:60-62 (1990); Rosenfeld et al., Science, 252:4341-4346 (1991); Morgan et al., Annu. Rev. Biochem., 62:191-217 (1993). Recombinant adenoviral vectors have the advantage over retroviruses of being able to transduce nonproliferating cells, as well as an ability to produce purified high titer virus.
In addition to viral-mediated gene delivery, a more recent means for DNA delivery has been receptor-mediated endocytosis. Endocytosis is the process by which eucaryotic cells continually ingest segments of the plasma membrane in the form of small endocytotic vesicles. Alberts et al., Mol. Biol. Cell, Garland Publishing Co., New York, 1983. Extracellular fluid and material dissolved in it becomes trapped in the vesicle and is ingested into the cell. Id. This process of bulk fluid-phase endocytosis can be visualized and quantified using a tracer such as enzyme peroxidase introduced into the extracellular fluid. Id.
Taking advantage of receptor-mediated endocytosis, the asialoglycoprotein receptor has been used in targeting DNA to HepG2 cells in vitro and liver cells in vivo. Wu et al., J. Biol. Chem., 262:4429-4432 (1987); Wu et al., Bio., 27:887-892 (1988); Wu et al., J. Biol. Chem., 263:14620-14624 (1988); Wu et al., J. Biol. Chem., 264:16985-16987 (1989); Wu et al., J. Biol. Chem., 266:14338-14342 (1991). These studies used asialoorosomucoid covalently linked to polylysine with water soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide or with 3xe2x80x2(2xe2x80x2pyridyl-dithio)propionic acid n-hydroxysuccinimide ester. Polylysine in the studies above bound DNA through ionic interaction. The DNA was ingested by endocytosis.
Other studies have utilized transferrin and the transferrin receptor for delivery of DNA to cells in vitro. Wagner et al., P.N.A.S., 87:3410-3414 (1990). These studies modified transferrin by covalently coupling transferrin to polylysine. Id. The polylysine interacted ionically with DNA. Delivery of DNA occurred to cells through the transferrin receptor. Such analyses were performed in vitro. Id. Cotten et al., P.N.A.S., 87:4033-4037 (1990); Zenk et al., P.N.A.S., 87:3655-3659 (1990).
In addition to delivery DNA (Gottschalk et al.,Gene Therapy 1:185-91 (1993)), other macromolecules can also be delivered by receptor-ligand systems. Leamon et al., P.N.A.S., 88:5572-5576 (1991); Leamon et al., J. Biol. Chem., 267:24966-24971(1992). In particular these studies have involved the folate receptor, an anchored glycosyl-phosphatidyl protein, which is excluded from coated pits and cycles in and out of the cells by caveolae. Anderson et al., Science, 252:410-411 (1992). This uptake mechanism has been called potocytosis. Id. Folate conjugated enzymes have been delivered into cells through this receptor system and retained activity for at least six hours. Leamon et al., P.N.A.S., 88:5572-5576 (1991). Folate receptors have limited tissue distribution and are overexpressed in several malignant cell lines derived from many tissues. Weitman et al., Cancer Res., 52:3396-3401 (1992); Weitman et al., Cancer Res., 52:6708-6711 (1992); Campbell, Cancer Res., 51:5329-5338 (1991); Coney, Cancer Res., 51:6125-6123 (1991). Other studies have also used biotin or folate conjugated to proteins by biotinylation for protein delivery to the cell. Low et al., U.S. Pat. No. 5,108,921.
Nucleic Acid Transporters for Delivery of Nucleic Acids into a Cell; Smith et al., U.S. patent application Ser. No. 08/484,777, filed Dec. 18, 1995, incorporated herein by reference in its entirety including any drawings.
A non-exchangeable apolipoprotein E peptide that mediates binding to the low density lipoprotein receptor is described in Mims et al., Journal of Biological Chemistry, 269 (32) 20539-20547, 1994, incorporated herein by reference in its entirety, including any drawings.
The present invention provides novel uses of lipophilic peptides for delivering macromolecules (e.g. nucleic acids) into a cell, complexes formed between the macromolecules to be delivered and the lipophilic peptide, and cells transformed by such complexes. Thus, the present invention allows for enhanced delivery of macromolecules (including nucleic acids) into cells.
The lipophilic peptide has a delivery peptide portion and a lipid moiety portion. The amino acid sequences of several suitable delivery peptides are set forth herein and those skilled in the art would be able to make and use many others given the methods described herein. The lipid moiety makes the delivery peptide lipophilic and examples of suitable modifications are provided herein. Again, however, those skilled in the art would be able to make and use lipophilic peptides having different lipid moieties.
Thus, in a first aspect, the present invention features a peptide-macromolecule complex for delivering a macromolecule into a cell. The complex includes a lipophilic peptide having a delivery peptide associated with a lipid moiety. The delivery peptide portion of the lipophilic peptide is complexed to the macromolecule.
The term xe2x80x9cpeptide-macromolecule complexxe2x80x9d as used herein refers to a molecular complex which is capable of transporting a macromolecule through the cell membrane. This molecular complex is preferably bound to a macromolecule noncovalently. The peptide-macromolecule complex should be capable of transporting nucleic acid in a stable and condensed state and of releasing the noncovalently bound nucleic acid into the cellular interior. Furthermore, the nucleic acid carrier may prevent lysosomal degradation of the nucleic acid by endosomal lysis. In addition, although not necessary, the peptide-macromolecule complex can also efficiently transport the nucleic acid through the nuclear membrane, as discussed below.
The peptide-macromolecule complex as described herein can contain, but is not limited to, seven components. It comprises, consists or consists essentially of: (1) a nucleic acid or other macromolecule with a known primary sequence that contains the genetic information of interest or a known chemical composition; (2) a peptide agent capable of stabilizing and condensing the nucleic acid or macromolecule in (1) above; (3) an N termini acylation moiety to increase the lipophilicity of the peptide agent in (2) above (4) a lysis moiety that enables the transport of the entire complex from the cell surface directly into the cytoplasm of the cell; (5) a moiety that recognizes and binds to a cell surface receptor or antigen or is capable of entering a cell through cytosis; (6) a moiety that is capable of moving or initiating movement through a nuclear membrane; and/or (7) a nucleic acid or macromolecular molecule binding moiety capable of covalently binding the moieties of (2), (3), (4), (5), and (6) above.
The term xe2x80x9cdeliveryxe2x80x9d refers to transportation of a molecule to a desired cell or any cell. Delivery can be to the cell surface, cell membrane, cell endosome, within the cell membrane, nucleus or within the nucleus, or any other desired area of the cell. Delivery includes not only transporting nucleic acid but also other macromolecules including, but not limited to, proteins, lipids, carbohydrates and various other molecules.
The term xe2x80x9cmacromoleculexe2x80x9d, refers to any natural and/or synthetic polymeric molecule capable of being in a biological environment and includes but is not limited to, proteins, oligonucleotides, dextrans, lipids or carbohydrates that can be delivered using the complexes or carrier systems described herein. The term xe2x80x9cnucleic acidxe2x80x9d as used herein refers to DNA or RNA. This would include naked DNA, a nucleic acid cassette, naked RNA, or nucleic acid contained in vectors or viruses. These are only examples and are not meant to be limiting.
A variety of proteins and polypeptides can be encoded by the nucleic acid. Those proteins or polypeptides which can be expressed include hormones, growth factors, enzymes, clotting factors, apolipoproteins, receptors, drugs, oncogenes, tumor antigens, tumor suppressors, cytokines, viral antigens, parasitic antigens, bacterial antigens and chemically synthesized polymers and polymers biosynthesized and/or modified by chemical, cellular and/or enzymatic processes. Specific examples of these compounds include proinsulin, insulin, growth hormone, androgen receptors, insulin-like growth factor I, insulin-like growth factor II, insulin growth factor binding proteins, epidermal growth factor, TGF-xcex1, TGF-xcex2, dermal growth factor (PDGF), angiogenesis factors (acidic fibroblast growth factor, basic fibroblast growth factor and angiogenin), matrix proteins (Type IV collagen, Type VII collagen, laminin), oncogenes (ras, fos, myc, erb, src, sis, jun), E6 or E7 transforming sequence, p53 protein, cytokine receptor, IL-1, IL-6, IL-8, IL-2, xcex1, xcex2, or xcex3IFN, GMCSF, GCSF, viral capsid protein, and proteins from viral, bacterial and parasitic organisms. Other specific proteins or polypeptides which can be expressed include: phenylalanine hydroxylase, xcex1-1-antitrypsin, cholesterol-7xcex1-hydroxylase, truncated apolipoprotein B, lipoprotein lipase, apolipoprotein E, apolipoprotein A1, LDL receptor, molecular variants of each, and combinations thereof. One skilled in the art readily appreciates that these proteins belong to a wide variety of classes of proteins, and that other proteins within these classes can also be used. These are only examples and are not meant to be limiting in any way.
It should also be noted that the genetic material which is incorporated into the cells from the above peptide-macromolecule complex includes (1) nucleic acid not normally found in the cells; (2) nucleic acid which is normally found in the cells but not expressed at physiological significant levels; (3) nucleic acid normally found in the cells and normally expressed at physiological desired levels; (4) other nucleic acid which can be modified for expression in cells; and (5) any combination of the above.
The term xe2x80x9clipophilic peptidexe2x80x9d as used herein refers to a peptide which is capable of stabilizing and condensing nucleic acid or a molecule, compound, or protein capable of achieving the same or substantially similar functional characteristics. This will include, but is not limited to, components which are capable of stabilizing and/or condensing nucleic acid by electrostatic binding, hydrophobic binding, hydrogen binding, intercalation or forming helical structures with the nucleic acid, including interaction in the major and/or minor grove of DNA. The term lipophilic peptides can also be referred herein as condensing agent. The lipophilic peptide is capable of noncovalently binding to nucleic acid. The lipophilic peptide is also capable of associating with a surface ligand, a nuclear ligand, and/or a lysis agent. Lipophilic peptides preferably refers to any peptide whose affinity for lipid surfaces is measured by a dissociation constant of Kdxcx9c10xe2x88x926 or less and whose xcex1-helicity is xcx9c55% in the presence of lipid. Non-lipophilic peptides have an affinity for lipid surfaces measured by a dissociation constant of  greater than Kd10xe2x88x925 and xcex1-helicity of  less than 40% in the presence of lipid. The term xe2x80x9cnon-exchangeble lipophilic peptidexe2x80x9d as used herein refers to any lipophilic peptide whose affinity for lipid surfaces is measured by a dissociation constant of xe2x89xa6Kd10xe2x88x929 and whose xcex1-helicity is xcx9c78% in the presence of lipid. The term xe2x80x9cxcex1-helicityxe2x80x9d as used herein refers to the preservation of the xcex1-helix conformation of the N terminal domain of the derivatized peptide in the presence of lipid.
In general, parameters that are important for lipophilic peptides include the following. First, the peptide must contain sufficient lysine or arginine residues to permit ionic interaction with the DNA. Second, the peptide must have sufficient length to form a stable helix, eleven or twelve residues, and condense the DNA to small particles, e.g., K8 forms larger particles than apoE3. Third, the peptide helix that forms upon interaction with DNA can be stabilized by leucine zipper formation which gives a condensing agent less susceptible to ionic strength. Finally, the lysine or arginine sequence of the condensing peptide serves as an additional function as a nuclear localization sequence.
By xe2x80x9cdelivery peptidexe2x80x9d is meant any amino acid sequence capable of transporting the macromolecule to the desired location in the body when the delivery peptide is associated with a lipid moiety. In the present invention, the most preferred delivery peptide sequence is found within the peptide apoE-3129-169 at residues 142-150. The amino acid sequence of this domain is RKLRKRLLR SEQ ID NO:1. In another preferred embodiment, the lipohilic peptide binding molecule is any peptide with the formula K(K)nVTK, SEQ ID NO:2 to SEQ ID NO:38 where n is 4, 5, 6, 7, 8 and homologues to n is 40. In another preferred embodiment, the lipophilic peptides is any peptide with the formula K(K)nXK, SEQ ID NO:39 to SEQ ID NO:75 where n is 4, 5, 6, 7, 8, and homologues to n is 40 where X is any naturally occurring amino acid and analogues thereof. In preferred embodiments the delivery peptide comprises, consists essentially of, or consists of a sequence set forth below or a functional fragment thereof:
STEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREG, SEQ ID NO:76
KKQLKKQLKKQLKQWK, SEQ ID NO:77
KKSPKKSPKKSPKKSWK, SEQ ID NO:78
KRRRRRRRRWR, SEQ ID NO:79
KLSKLEKKWSKLEK, SEQ ID NO:80
KLSKLEKKLSKLEKKWSKLEK, SEQ ID NO:81
KSLKKSLKKSLKKSWK, SEQ ID NO:82
KSTPPKKKRKVEDPKDFPSELLSA, SEQ ID NO:83
KAKKKK-NH-(CH2)2-SS-(CH2)2-CO-KKKKWK, SEQ ID NO:84
KIRRRGKNKVAARTCRQRRTDR, SEQ ID NO:85
KXKKXKKKXKKXKWK, (where X is A or S) SEQ ID NO:86
KIRRRGKNKAAARTCRERRRSK, SEQ ID NO:87
KIRRRGKNKVAAQNCRKRKLDQ, SEQ ID NO:88
KIRRRGKNKVAAQNCRKRKLET, SEQ ID NO:89
KRRIRREKNKMAAAKCRNRRRELT, SEQ ID NO:90
GRPRAINKHEQEQISRLLEKGHPRQQLAIIFGIGVSTLYRYFPASSIKKRMN, SEQ ID NO:91
KSGPRPRGTRGKGRRIRR, SEQ ID NO:92
KDRSNLLERHTR, SEQ ID NO:93
KRPAATKKAGQAKKKL, SEQ ID NO:94
K(K)nWK, SEQ ID NO:95 to SEQ ID NO:131 where n is 4, 5, 6, 7, 8 and homologues to n is 40,
K(K)nXK, SEQ ID NO:39 to SEQ ID NO:75 where n is 4, 5, 6, 7, 8, and homologues to n is 40
where X is any naturally occurring amino acid and analogues thereof,
KSPLLKSMKGIKQQQHP-(SPNQQQHP)nGK, SEQ ID NO:132 to SEQ ID NO:137 where n is 1-6.
This would include the use of any subfragments of the above which provide nucleic acid stability and condensing characteristics. Furthermore, this would include any derivatives, analogs or modifications of the above peptides. The above peptides can include lysine or arginine residues for electrostatic binding to nucleic acid. These positively charged amino acids help hold the nucleic acid intact. Other examples include or onrnithine, homolysine, homoarginine and 2,4-diaminobutyric acid. The lipophilic peptides can also contain tyrosine which is useful in determining peptide concentration and iodination for tracking purposes in vitro and in vivo. Tryptophan also increases the stability of interaction with the nucleic acid through intercalation. In addition, binding of the peptide to DNA quenches tryptophan fluorescence and allows the kinetics and thermodynamics of complex formation to be determined. The lipophilic peptides can also contain helix forming residues such as tryptophan, alanine, leucine or glutamine. These can act as spacers which allow the cationic residues to adopt an optimal configuration for interaction with the nucleic acid in a helical manner, resulting in a more stable complex. Furthermore, the lipophilic peptides can also include a stabilized cyclic version of any of the above mentioned peptides. Such a cyclic version can be formed by introducing a lactam or disulfide bridge. Likewise, dimers of any of the above mentioned peptides can also be used as a binding moiety.
The term delivery peptide can also encompass any derivatives or peptidomimetics in which the peptide bond or backbone of the peptide has been replaced with a molecular skeleton so that the functional residues of the peptide are preserved, and conformationally constrained, in approximately the correct positions for interaction with the active sites on the original peptide. This substitution may include but is not restricted to any atoms of the delivery peptide such as the O and NH atoms of the peptide backbone or other atoms which are rarely involved in close interactions with the active site. xe2x80x9cPeptidomimeticsxe2x80x9d may encompass any such substitutions resulting in the preservation of the residue interactions which are paramount in the proper functioning of the peptide. For example, one peptidomimetic resulted from the substitution of a non-peptidal architectural spacer (i.e., a benzodiazepine-based xcex2-turn mimetic) so that the functional side-chain residues were positioned so that their C-xcex1 atoms could occupy equivalent positions to those occupied in the native peptide. This example is not meant to be limiting. Any such peptidomimetics or analogues are encompassed herein.
By xe2x80x9clipid moietyxe2x80x9d is meant any agent capable of attaching to a delivery peptide and that upon attachment imparts lipophilic qualities to the overall lipophilic peptide. This preferably increases the lipophilicity of the delivery peptide. Lipophilicity is measured by the decrease in the dissociation constant of the molecule (Kd) after attachment. In preferred embodiments, the lipid moiety is a distearyl derivative selected from the group consisting of: (1) N,N-distearyl-glycyl-; (2) "xgr"-N,N-distearylglycyl-; and (3) N,N-distearylamidomethyl. Other embodiments of the lipid moiety are straight chain or branched chain alkyl groups containing either 0, 1, 2 to 6 unsaturated bonds, with chain lengths from 6 to 30 carbon atoms. Alternatively, the lipid moiety is a dipalmitoyl derivative selected from the group consisting of: Nxcex1, Nxcex5xe2x80x2-dipalmitoyl-, and Nxcex1, Nxcex5-dipaimitoyl. Other embodiments of the lipid moiety are straight chain or branched chain alkyl groups containing either 0, 1, 2 to 6 unsaturated bonds, with chain lengths from 6 to 30 carbon atoms.
The complex is preferably isolated, purified, or enriched. Thus, the complex or carrier system is present in a state that is not found in nature and that is not possible without human intervention. In other preferred embodiments, the complex is capable of binding with a cell surface receptor, lysing an endosome, and targeting the nucleus of said cell. In order to achieve these functions, the lipophilic peptide and/or lipophilic moiety may be associated with a lysis agent, a surface ligand or a nuclear ligand.
The term xe2x80x9cassociated withxe2x80x9d as used herein refers to binding, attaching, connecting or linking molecules through covalent means or noncovalent means. xe2x80x9cAssociated withxe2x80x9d includes, but is not limited to, a lipophilic peptides associated with a surface ligand, nuclear ligand and/or a lysis agent. In addition, it includes the association of a spacer (discussed below) with the above components.
The term xe2x80x9clysis agentxe2x80x9d as used herein refers to a molecule, compound, protein or peptide which is capable of breaking down an endosomal membrane and freeing the contents into the cytoplasm of the cell. The lysis agent can work by: (1) a membrane fusion mechanism, i.e., fusogenic, whereby the lysis agent associates or fuses with the cell membrane to allow the endosomal contents to leak into the cytoplasm; (2) a membrane destabilization mechanism whereby the lysis agent disrupts the structural organization of the cell membrane thereby causing leakage through the endosome into the cytoplasm; or (3) other known or unknown mechanisms which cause endosomal lysis. This term includes, but is not limited to, synthetic compounds such as the JTS-1 peptide, viruses, lytic peptides, or derivatives thereof. The term xe2x80x9clytic peptidexe2x80x9d refers to a chemical grouping which penetrates a membrane such that the structural organization and integrity of the membrane is lost. As a result of the presence of the lysis agent, the membrane undergoes lysis, fusion or both.
In the present invention, a preferred lysis agent is the JTS-1 peptide or derivatives thereof. The amino acid sequence of JTS-1 lytic peptide is GLFEALLELLESLWELLLEA. SEQ ID NO:138. One skilled in the art will readily appreciate and understand that such nomenclature is the standard notation accepted in the art for designating amino acids. The JTS-1 lytic peptide and derivatives are designed as an xcex1-helix, which contains a sequence of amino acids such that the side chains are distributed to yield a peptide with hydrophobic and hydrophilic sides. Such xcex1-helixes are termed amphipathic or amphiphilic. The hydrophobic side contains highly apolar amino acid side chains, both neutral and non-neutral. The hydrophilic side contains an extensive number of glutamic acids but could also contain aspartic acid, as well as polar or basic amino acids. The JTS-1 peptide would include any derivatives or modifications of the backbone thereof. The lytic peptide undergoes secondary structure changes at acidic pH resulting in the formation of oligomeric aggregates which possess selective lytic properties.
In general, parameters that are important for amphiphilic peptide lysis activity include the following. First, Hydrophobicity: The peptide must have a high enough hydrophobicity of the hydrophobic face to interact with and penetrate phospholipid-cholesterol membranes, i.e., lipid binding per se is not sufficient. Red cell hemolysis assays give better indications of which peptides will have useful activity. Second, Peptide aggregation: The ability to aggregate plays an important role in lysis and transfection. Third, pH sensitivity: The amphiphilic peptide must be pH sensitive. Lysis activity can be controlled by the introduction of lysine, arginine and histidine residues into the hydrophilic face of JTS-1. Fourth, Lipid membrane interaction: The peptide must have a hydrophobic carboxyl terminal to permit interaction with lipid membranes, e.g., tyrosine substitution for tryptophan greatly reduces activity. Finally, Peptide chain length: The length must be greater than twelve residues in order to get stable helix formation and lipid membrane penetration and rupture.
Lytic Peptides, Analogs and Derivatives
In order to eliminate the use of adenovirus as an endosomal lysis agent, fusogenic or membrane disruptive peptides were designed which would increase the rate of delivery of nucleic acid from the endosome to the cell and ensure that higher concentrations of the endocytosed nucleic acid would be released and not degraded in the endosomes. In addition to pH sensitive liposomes (Liu and Haung,1990 Biochim Biophys Acta 1022:348-54), composed of phosphatidylsuccinylglycerol and phosphatidylethanolamine, lipophilic derivatives of GLFEALLELLESLWELLLEA SEQ ID NO:138 (JTS-1) and other lytic peptides were used. A number of fusogenic/lytic peptides have been previously described, including the amino terminal sequence of the vesicular stomatitis virus glycoprotein and the synthetic amphipathic peptide GALA. Ojcius et al., TIBS, 16:225-229 (1991); Doms et al., Membrane Fusion, pp. 313-335 (Marcel Dekker, Inc., N.Y. 1991); Subbarao et al., Biochemistry, 26:2964-2972 (1987).
Short synthetic peptides from the hemagglutinin HA2 subunit of influenza have been studied with artificial lipid membranes. Wharton et al., J. Gen. Virol., 69:1847-1857 (1988). These peptides give both membrane fusion and leakage of liposomal contents similar to whole hemagglutinin molecules. However, the rates are quite slower.
In order to increase the low efficiency rate by endosomal lysis with influenza peptides, new peptides were created. In creating these new peptides for endosomal lysis, four factors were considered: (1) the content and spacing of the hydrophilic and hydrophobic amino acid residues along the xcex1-helix to direct organized oligomer association of the peptides after their insertion into the membrane; (2) covalent attachment of the peptide to a binding molecule and preclusion of oligomer formation and the necessary aggregation; (3) sufficient aggregation of several oligomeric structures necessary to achieve lysis; and (4) presence of hydrophilic carboxyl and amino side chain and terminal groups to create the pH sensitive endosomal processing.
It is well known that the distribution of the amino acid side chains along the peptide chain determines the secondary and tertiary structure of a protein. For membrane associating proteins, the amphipathic profile created by the hydrophobic and hydrophilic residues is a principal determinant of the function of the protein. Analysis of the region of the influenza hemagglutinin responsible for fusion of the viral envelope with the plasma membrane of cells reveals that a large hydrophobic surface is formed when the protein becomes xcex1-helical.
In the present invention, a number of lytic peptides have been designed and tested for endosomal lytic activity. In order for these peptides to be functional, they must have the following parameters. These peptides are amphipathic membrane associating peptides. These amphipathic peptides were designed as an xcex1-helix, containing a sequence of amino acids such that the side chains are distributed so that the peptide has a hydrophobic and hydrophilic side. The hydrophobic side contains highly apolar amino acid side chains, while the hydrophilic side contains an extensive number of glutamic acids.
In general, the amphipathic membrane associating peptides usually contain 21 amino acids or fewer. The design criteria requires that the amino acids have a high probability of forming amphiphilic species. This can be exhibited in the secondary structure of the membrane associating peptides, i.e., helices, turns, bends, loops, xcex2-sheets, and their oligomeric aggregates and other super secondary structures defined in the literature, e.g., helix-turn-helix. In addition, the amino acids should have a high probability of being found in an xcex1-helix and a low probability of forming a xcex2-sheet or turn structure. Leucine, lysine and glutamate are appropriate amino acids for such characteristics. For example, lysine positioned on the lateral face of the xcex1-helix and glutamate residues opposite leucine provide optimal charge distribution for lipid interaction. Furthermore, lysines and glutamates can be positioned to take advantage of potential helix stabilization. Helix dipole stabilization is optimized by removing the charge at the NH2 and COOH-termini so NH2 termini and COOH-terminal amides are useful. Such probabilities can be determined from secondary structural predictions or analogous methods to optimize secondary structural design. Unnatural amino acid which have been described for their propensity to induce helix structures in peptides are also used.
The hydrophobic or lipophilic face has a great effect on lipid-peptide interactions. Thus, the lipophilic face is modeled after peptides known to interact with lipids. Hydrophobic and lipid interactive residues (Ala, Leu, Met, Val, Phe, Trp, Tyr, Cys, Pro) when substituted on the lipophilic face either singularly or collectively promote a similar membrane associating effect. Similarly, an acid group and/or hydrophilic group (Glu, Gln, His, Lys, Gly, Ser, Asp, Asn, Pro, Arg) can be placed on the hydrophilic face to achieve the objective. The lipophilic and hydrophilic faces can also contain residues which promote lipid interaction and/or induce endosomal lysis at acidic pH. Such an interaction is not limited to an xcex1-helix promoting residue since glycine and serine positioned on the hydrophilic face have been shown to favorably influence activity as seen with the examples below.
One in particular, the JTS-1 peptide, GLFEALLELLESLWELLLEA, SEQ ID NO:138 has a hydrophobic face which contains only strongly apolar amino acids, while the hydrophilic face is dominated by negatively charged glutamic acid residues at physiological pH values. At the amino terminus end, the JTS-1 peptide uses the Gly-Leu-Phe sequence at amino acid positions 1-2-3, respectively, as a fusogenic or membrane disruptive sequence. For increased pH sensitivity Glu is added at amino acid position 4. In addition, at positions 12-15, Ser-Leu-Trp-Glu is used as a lipid binding site. The remaining sequences are arranged to provide the hydrophobic and hydrophilic face of apoE-3. Amino acids 16, 9, 2, 13, 6, 17, 10, 3, 14, 7 and 18 form the hydrophobic face. Amino acids 5, 12, 1, 8, 15, 4 and 11 form the hydrophilic face.
The following JTS peptide was characterized for lytic activity: apoE-3 GLFEALLELLESLWELLLEA SEQ ID NO:138.
In addition to the above, n-acyl tetrapeptides with fusogenic or membrane destabilizing activity can be constructed. The structure of these is set forth in Smith et al., U.S. patent application Ser. No. 08/484,777, filed Dec. 18, 1995, incorporated herein by reference in its entirety including any drawings. The tetrapeptide sequence when substituted with the appropriate amino acids as discussed above are capable of interacting with lipid bilayers and thereby destabilizing. The acyl chain can be lengthened or shortened depending on structure/function requirements.
Furthermore, shorter xcex1-helical peptides were also synthesized with the above design motifs in mind to retain the lytic properties as discussed above. Furthermore, a COOH-terminal amide is used to provide helix-dipole optimization. When in an xcex1-helical structure the hydrophobic face appears at positions 4, 7, 3, and 10.
To provide the Gly-Leu-Phe fusogenic or membrane disruption activity to the above-mentioned xcex1-helical peptide the peptide was lengthened to an 11-mer. Adding the additional amino acid to form the following peptide, Suc-GLFKLLEEWLE, SEQ ID NO:139 allowed the activity of the three glutamic acids to be retained. In addition, the peptide was succinylated at the amino terminus to afford an i to i+4 salt bridge with lysine which is designed to stabilize the helix.
Additional detailed descriptions of other lysis agents are provided in Smith et al., U.S. patent application Ser. No. 08/484,777, filed Dec. 18, 1995, incorporated herein by reference in its entirety including any drawings.
The term xe2x80x9csurface ligandxe2x80x9d as used herein refers to a chemical compound or structure which will bind to a surface receptor of a cell. The term xe2x80x9ccell surface receptorxe2x80x9d as used herein refers to a specific chemical grouping on the surface of a cell to which the ligand can attach. Cell surface receptors can be specific for a particular cell, i.e., found predominantly in one cell rather than in another type of cell (e.g., LDL and asialoglycoprotein receptors are specific for hepatocytes). The receptor facilitates the internalization of the ligand and attached molecules. A cell surface receptor includes, but is not limited to, a folate receptor, biotin receptor, lipoic acid receptor, low-density lipoprotein receptor, asialoglycoprotein receptor, insulin-like growth factor type II/cation-independent mannose-6-phosphate receptor, calcitonin gene-related peptide receptor, insulin-like growth factor I receptor, nicotinic acetylcholine receptor, hepatocyte growth factor receptor, endothelin receptor, bile acid receptor, bone morphogenetic protein receptor, cartilage induction factor receptor or glycosylphosphatidylinositol (GPI)-anchored proteins (e.g., xcex2-andrenargic receptor, T-cell activating protein, Thy-1 protein, GPI-anchored 5xe2x80x2 nucleotidase). These are nonlimiting examples.
A receptor is a molecule to which a ligand binds specifically and with relatively high affinity. It is usually a protein or a glycoprotein, but may also be a glycolipid, a lipidpolysaccharide, a glycosaminoglycan or a glycocalyx. For purposes of this invention, epitopes to which an antibody or its fragments binds is construed as a receptor since the antigen:antibody complex undergoes endocytosis. Furthermore, surface ligand includes anything which is capable of entering the cell through cytosis (e.g., endocytosis, potocytosis, pinocytosis).
As used herein, the term xe2x80x9cligandxe2x80x9d refers to a chemical compound or structure which will bind to a receptor. This includes but is not limited to ligands such as asialoorosomucoid, asialoglycoprotein, folate, lipoic acid, biotin, as well as those compounds listed in PCT publication WO 93/18759, hereby incorporated by reference including all drawings, sketches or diagrams.
One skilled in the art will readily recognize that the ligand chosen will depend on which receptor is being bound. Since different types of cells have different receptors, this provides a method of targeting nucleic acid to specific cell types, depending on which cell surface ligand is used. Thus, the preferred cell surface ligand may depend on the targeted cell type.
The term xe2x80x9cnuclear ligandxe2x80x9d as used herein refers to a ligand which will bind a nuclear receptor. The term xe2x80x9cnuclear receptorxe2x80x9d as used herein refers to a chemical grouping on the nuclear membrane which will bind a specific ligand and help transport the ligand through the nuclear membrane. Nuclear receptors can be, but are not limited to, those receptors which bind nuclear localization sequences. Nonlimiting examples of nuclear ligands include those disclosed in PCT publication WO 93/18759, hereby incorporated by reference including all drawings, sketches, diagrams and illustrations.
As noted above, the surface ligand, the nuclear ligand and/or the lysis agent can be associated directly to the lipohilic peptide binding molecule or can be associated with the lipohilic peptide binding molecule via a spacer. Such as those described in Smith et al., supra, incorporated herein.
The macromolecule, as noted above, may be nucleic acid, such as DNA or RNA. The lipid moiety may be linked to the N-terminus of said delivery peptide and the delivery peptide may be non-covalently bound to said macromolecule. The macromolecule may be complexed with more than one lipophilic peptides, for example, with two, three, four, or five lipophilic peptides. The functional characteristics described above (i.e., condensation, lysis, nuclear targeting, etc.)can be separately performed from different peptide-macromolecule complexes. The delivery peptide may comprise a compound selected from the group consisting of: (1) apoE-3129-169; (2) apoE-3139-169; and (3) apoE-3129-169Q142 as described in Mims et al., Jour. Biol. Chem. 269, 20539-20547.
In another aspect, the invention features a method of using a complex described above for delivering said macromolecule to a cell comprising the step of contacting said cell with said complex for a time sufficient to permit incorporation of said complex into said cell, wherein said macromolecule is delivered in a physiologically sufficient amount.
In preferred embodiments, the method also involves contacting said complex with a biological detergent capable of solubilizing and/or enmeshing said macromolecule. The term xe2x80x9cenmeshedxe2x80x9d as used herein refers to the covering, complexing or association of the delivery peptide with a nucleic acid macromolecule resulting in the condensation of the peptide-macromolecule complex. The detergentmay be selected from the group consisting of: CHAPS (N,N-dimethyl-N-(3-sulfopropyl)-3-[[3xcex1,5xcex2,7xcex1,12xcex1)-3,7,12-trihydroxy-24-oxocholan-24-yl]amino]-1-propanaminium hydroxide inner salt), 1-O-octyl-D-glucoside and other zwitterionic and neutral detergents except sodium cholate. The detergent preferably is present at a final concentration which is below the critical micelle concentration (cmc)of the detergent. The term xe2x80x9ccritical micelle concentrationxe2x80x9d refers to that concentration of detergent below which formation of micellar structures is promoted. In addition, the detergent preferably has a dilution ratio with said macromolecule sufficient to reduce the concentration of said detergent below its critical micelle concentration.
In another aspect, the invention features a cell transformed with a complex described above. As used herein xe2x80x9ctransformationxe2x80x9d or xe2x80x9ctransformedxe2x80x9d is a mechanism of gene transfer which involves the uptake of nucleic acid by a cell or organism. It is a process or mechanism of inducing transient or permanent changes in the characteristics (expressed phenotype) of a cell. Such changes are by a mechanism of gene transfer whereby DNA or RNA is introduced into a cell in a form where it expresses a specific gene product or alters the expression or effect of endogenous gene products. Following entry into the cell, the transforming nucleic acid may recombine with that of the host. Such transformation is considered stable transformation in that the introduction of gene(s) into the chromosome of the targeted cell where it integrates and becomes a permanent component of the genetic material in that cell. Gene expression after stable transformation can permanently alter the characteristics of the cell leading to stable transformation. In addition, the transforming nucleic acid may exist independently as a plasmid or a temperate phage, or by episomes. An episomal transformation is a variant of stable transformation in which the introduced gene is not incorporated in the host cell chromosomes but rather remains in a transcriptionally active state as an extrachromosomal element.
Transformation can be performed by in vivo techniques as described below, or by ex vivo techniques in which cells are cotransfected with a peptide-macromolecule complex containing nucleic acid and also containing a selectable marker. This selectable marker is used to select those cells which have become transformed. It is well known to those skilled in the art the type of selectable markers to be used with transformation studies.
The transformed cells can produce a variety of compounds selected from proteins, polypeptides or RNA, including hormones, growth factors, enzymes, clotting factors, apolipoproteins, receptors, drugs, tumor antigens, viral antigens, parasitic antigens, and bacterial antigens. Other examples can be found above in the discussion of nucleic acid. The product expressed by the transformed cell depends on the nucleic acid used. The above are only examples and are not meant to be limiting.
These methods of use would include the steps of contacting a cell with a peptide-macromolecule complex as described above for a sufficient time to transform the cell. Cell types of interest can include, but are not limited to, liver, muscle, lung, endothelium, bone, blood, joints and skin.
Other features and advantages of the invention will be apparent from the following detailed description of the invention in conjunction with the accompanying drawings and from the claims.